Project Summary ?-Secretase is a membrane-embedded protease complex with presenilin as its catalytic component. This complex cleaves the transmembrane domains (TMDs) of a wide variety of type I integral membrane proteins after their ectodomain release by sheddases. Among these substrates are Notch and the amyloid ?-protein precursor (APP). TMD cleavage of Notch receptors is part of cell signaling mechanisms essential to metazoan biology and dysregulated in cancer, and TMD cleavage of APP to amyloid ?-protein (A?) is essential to the pathogenesis of Alzheimer's disease (AD). The overarching goal of this proposal is to elucidate how intramembrane proteolysis is accomplished by ?-secretase. Toward this end, we will address three central open questions regarding the mechanism of this complex enzyme that is so critical in biology and medicine. (1) How does ?-secretase recognize substrates? As the enzyme cleaves the TMD of substrate, we seek to understand the role of helicity in substrate recognition. In addition, we have used a recent high-resolution structure of the protease complex to design experiments to identify the gate on presenilin that allows access of substrate TMD into the active site. (2) How does ?-secretase carry out processive proteolysis? ?-Secretase initially cleaves substrates within their TMDs close to the cytosolic interface, followed by processive carboxypeptidase trimming resulting in secreted peptides. We will address whether the longer A? peptide intermediates have higher affinity for the enzyme, allowing more time for trimming, than shorter A? peptides. We will also address whether peptide product intermediates remain bound or dissociate from the enzyme prior to further trimming. (3) How does ?-secretase unwind helical TMD substrates for proteolysis? We have designed a series of hybrid helical peptide/transition-state analog inhibitors to mimic the TMD substrate bound to the enzyme in the transition state. In collaboration with a leading structural biology lab, structure elucidation of the most potent hybrid inhibitor bound to active ?-secretase will be determined to understand the structural basis of the ?-secretase mechanism of action.